RESUMO
People living with HIV (PLH) have significantly higher rates of cognitive impairment (CI) and major depressive disorder (MDD) versus the general population. The enzyme neutral sphingomyelinase 2 (nSMase2) is involved in the biogenesis of ceramide and extracellular vesicles (EVs), both of which are dysregulated in PLH, CI, and MDD. Here we evaluated EcoHIV-infected mice for behavioral abnormalities relevant to depression and cognition deficits, and assessed the behavioral and biochemical effects of nSMase2 inhibition. Mice were infected with EcoHIV and daily treatment with either vehicle or the nSMase2 inhibitor (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-carbamate (PDDC) began 3 weeks post-infection. After 2 weeks of treatment, mice were subjected to behavior tests. EcoHIV-infected mice exhibited behavioral abnormalities relevant to MDD and CI that were reversed by PDDC treatment. EcoHIV infection significantly increased cortical brain nSMase2 activity, resulting in trend changes in sphingomyelin and ceramide levels that were normalized by PDDC treatment. EcoHIV-infected mice also exhibited increased levels of brain-derived EVs and altered microRNA cargo, including miR-183-5p, miR-200c-3p, miR-200b-3p, and miR-429-3p, known to be associated with MDD and CI; all were normalized by PDDC. In conclusion, inhibition of nSMase2 represents a possible new therapeutic strategy for the treatment of HIV-associated CI and MDD.
Assuntos
Transtorno Depressivo Maior , Vesículas Extracelulares , Infecções por HIV , MicroRNAs , Animais , Ceramidas , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/farmacologia , Esfingomielina Fosfodiesterase/genéticaRESUMO
Suppression of HIV replication by antiretroviral therapy (ART) or host immunity can prevent AIDS but not other HIV-associated conditions including neurocognitive impairment (HIV-NCI). Pathogenesis in HIV-suppressed individuals has been attributed to reservoirs of latent-inducible virus in resting CD4+ T cells. Macrophages are persistently infected with HIV but their role as HIV reservoirs in vivo has not been fully explored. Here we show that infection of conventional mice with chimeric HIV, EcoHIV, reproduces physiological conditions for development of disease in people on ART including immunocompetence, stable suppression of HIV replication, persistence of integrated, replication-competent HIV in T cells and macrophages, and manifestation of learning and memory deficits in behavioral tests, termed here murine HIV-NCI. EcoHIV established latent reservoirs in CD4+ T lymphocytes in chronically-infected mice but could be induced by epigenetic modulators ex vivo and in mice. In contrast, macrophages expressed EcoHIV constitutively in mice for up to 16 months; murine leukemia virus (MLV), the donor of gp80 envelope in EcoHIV, did not infect macrophages. Both EcoHIV and MLV were found in brain tissue of infected mice but only EcoHIV induced NCI. Murine HIV-NCI was prevented by antiretroviral prophylaxis but once established neither persistent EcoHIV infection in mice nor NCI could be reversed by long-acting antiretroviral therapy. EcoHIV-infected, athymic mice were more permissive to virus replication in macrophages than were wild-type mice, suffered cognitive dysfunction, as well as increased numbers of monocytes and macrophages infiltrating the brain. Our results suggest an important role of HIV expressing macrophages in HIV neuropathogenesis in hosts with suppressed HIV replication.
Assuntos
Linfócitos T CD4-Positivos/virologia , Reservatórios de Doenças , Infecções por HIV/complicações , HIV/fisiologia , Macrófagos Peritoneais/virologia , Transtornos Neurocognitivos/virologia , Transferência Adotiva , Idoso , Animais , Antirretrovirais/uso terapêutico , Encéfalo/virologia , Feminino , HIV/genética , HIV/imunologia , HIV/patogenicidade , Infecções por HIV/tratamento farmacológico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Pessoa de Meia-Idade , Plasmídeos , Baço/citologia , Baço/imunologiaRESUMO
Cigarette smoke usage is prevalent in human immunodeficiency virus (HIV)-positive patients, and, despite highly active antiretroviral therapy, these individuals develop an accelerated form of chronic obstructive pulmonary disease (COPD). Studies investigating the mechanisms of COPD development in HIV have been limited by the lack of suitable mouse models. Here we describe a model of HIV-induced COPD in wild-type mice using EcoHIV, a chimeric HIV capable of establishing chronic infection in immunocompetent mice. A/J mice were infected with EcoHIV and subjected to whole body cigarette smoke exposure. EcoHIV was detected in alveolar macrophages of mice. Compared with uninfected mice, concomitant EcoHIV infection significantly reduced forced expiratory flow 50%/forced vital capacity and enhanced distal airspace enlargement following cigarette smoke exposure. Lung IL-6, granulocyte-macrophage colony-stimulating factor, neutrophil elastase, cathepsin G, and matrix metalloproteinase-9 expression was significantly enhanced in smoke-exposed EcoHIV-infected mice. These changes coincided with enhanced IκBα, ERK1/2, p38, and STAT3 phosphorylation and lung cell apoptosis. Thus, the EcoHIV smoke exposure mouse model reproduces several of the pathophysiological features of HIV-related COPD in humans, indicating that this murine model can be used to determine key parameters of HIV-related COPD and to test future therapies for this disorder.
Assuntos
Infecções por HIV/complicações , Doença Pulmonar Obstrutiva Crônica/complicações , Animais , Apoptose , Modelos Animais de Doenças , Humanos , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Macrófagos Alveolares/patologia , Macrófagos Alveolares/virologia , Masculino , Camundongos , Neutrófilos/metabolismo , Peptídeo Hidrolases/metabolismo , Pneumonia/patologia , Fumar/efeitos adversosRESUMO
BACKGROUND: EcoHIV is a chimeric HIV that replicates in mice in CD4+ T cells, macrophages, and microglia (but not in neurons), causing lasting neurocognitive impairment resembling neurocognitive disease in people living with HIV. The present study was designed to develop EcoHIV-susceptible primary mouse brain cultures to investigate the indirect effects of HIV infection on neuronal integrity. RESULTS: We used two EcoHIV clones encoding EGFP and mouse bone marrow-derived macrophages (BMM), mixed mouse brain cells, or enriched mouse glial cells from two wild-type mouse strains to test EcoHIV replication efficiency, the identity of productively infected cells, and neuronal apoptosis and integrity. EcoHIV replicated efficiently in BMM. In mixed brain cell cultures, EcoHIV targeted microglia but did not cause neuronal apoptosis. Instead, the productive infection of the microglia activated them and impaired synaptophysin expression, dendritic density, and axonal structure in the neurons. EcoHIV replication in the microglia and neuronal structural changes during infection were prevented by culture with an antiretroviral. CONCLUSIONS: In murine brain cell cultures, EcoHIV replication in the microglia is largely responsible for the aspects of neuronal dysfunction relevant to cognitive disease in infected mice and people living with HIV. These cultures provide a tool for further study of HIV neuropathogenesis and its control.
Assuntos
Encéfalo , Microglia , Neurônios , Replicação Viral , Animais , Camundongos , Encéfalo/virologia , Encéfalo/patologia , Neurônios/virologia , Neurônios/patologia , Microglia/virologia , Células Cultivadas , Infecções por HIV/virologia , Macrófagos/virologia , Modelos Animais de Doenças , Apoptose , Humanos , HIV-1/fisiologia , Cultura Primária de Células , Camundongos Endogâmicos C57BLRESUMO
HIV enters the brain within days of infection causing neurocognitive impairment (NCI) in up to half of infected people despite suppressive antiretroviral therapy. The virus is believed to enter the brain in infected monocytes through chemotaxis to the major monocyte chemokine, CCL2, but the roles of CCL2 in established NCI are not fully defined. We addressed this question during infection of conventional and CCL2 knockout mice with EcoHIV in which NCI can be verified in behavioral tests. EcoHIV enters mouse brain within 5 days of infection, but NCI develops gradually with established cognitive disease starting 25 days after infection. CCL2 knockout mice infected by intraperitoneal injection of virus failed to develop brain infection and NCI. However, when EcoHIV was directly injected into the brain, CCL2 knockout mice developed NCI. Knockout of CCL2 or its principal receptor, CCR2, slightly reduced macrophage infection in culture. Treatment of mice prior to and during EcoHIV infection with the CCL2 transcriptional inhibitor, bindarit, prevented brain infection and NCI and reduced macrophage infection. In contrast, bindarit treatment of mice 4 weeks after infection affected neither brain virus burden nor NCI. Based on these findings we propose that HIV enters the brain mainly through infected monocytes but that resident brain cells are sufficient to maintain NCI. These findings suggest that NCI therapy must act within the brain.
Assuntos
Complexo AIDS Demência , Quimiocina CCL2 , Infecções por HIV , Animais , Camundongos , Complexo AIDS Demência/genética , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Cognição , Infecções por HIV/complicações , Infecções por HIV/genética , Indazóis , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos , Receptores CCR2/genética , Modelos Animais de DoençasRESUMO
Thirty-eight million people worldwide are living with HIV, PWH, a major public health problem. Antiretroviral therapy (ART) revolutionized HIV treatment and significantly increased the lifespan of PWH. However, approximately 15-50% of PWH develop HIV associated neurocognitive disorders (HIV-NCI), a spectrum of cognitive deficits, that negatively impact quality of life. Many PWH also have opioid use disorder (OUD), and studies in animal models of HIV infection as well as in PWH suggest that OUD can contribute to HIV-NCI. The synthetic opioid agonist, buprenorphine, treats OUD but its effects on HIV-NCI are unclear. We reported that human mature inflammatory monocytes express the opioid receptors MOR and KOR, and that buprenorphine reduces important steps in monocyte transmigration. Monocytes also serve as HIV reservoirs despite effective ART, enter the brain, and contribute to HIV brain disease. Using EcoHIV infected mice, an established model of HIV infection and HIV-NCI, we previously showed that pretreatment of mice prior to EcoHIV infection reduces mouse monocyte entry into the brain and prevents NCI. Here we show that buprenorphine treatment of EcoHIV infected mice with already established chronic NCI completely reverses the disease. Disease reversal was associated with a significant reduction in brain inflammatory monocytes and reversal of dendritic injury in the cortex and hippocampus. These results suggest that HIV-NCI persistence may require a continuing influx of inflammatory monocytes into the brain. Thus, we recommend buprenorphine as a potential therapy for mitigation of HIV brain disease in PWH with or without OUD.
Assuntos
Encefalopatias , Buprenorfina , Infecções por HIV , Transtornos Relacionados ao Uso de Opioides , Animais , Humanos , Camundongos , Buprenorfina/farmacologia , Buprenorfina/uso terapêutico , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Analgésicos Opioides/uso terapêutico , Qualidade de Vida , Transtornos Relacionados ao Uso de Opioides/complicações , Receptores OpioidesRESUMO
The elevation of nitric oxide (NO) within the central nervous system (CNS) is known to be associated with the pathogenesis of neurodegenerative diseases such as HIV-associated dementia (HAD), brain ischemia, Parkinson's disease, and Alzheimer's disease. NO is enzymatically formed by the enzyme nitric oxide synthase (NOS). There are two forms of NOS, the constitutive and the inducible form. The constitutive form is present in endothelial cells (eNOS) and neurons (nNOS). The inducible form (iNOS) is expressed in various cell types including astroglia and microglia of the CNS. Using an animal model, we investigated the involvement of eNOS in the pathology of prion disease. We showed dramatic upregulation of eNOS immunoreactivity in reactive astroglial cells in the hippocampus in the prion disease animal model, scrapie in mice. Expression of eNOS was upregulated in cytosolic and mitochondrial fractions of whole brain. In the hippocampal region, eNOS was widely overexpressed in various components of the cell. We found that eNOS dramatically accumulated in hippocampal mitochondria and was particularly prevalent in structurally dysfunctional mitochondria. In association with the accumulation of eNOS in mitochondria, we showed that mitochondrial superoxide dismutase (Mn-SOD or SOD2), cytochrome c, and ATP activity were downregulated both in whole brain and in the hippocampal region. These results indicate that eNOS plays a role in the development of dysfunctional mitochondria and this, in turn, could induce some of the histopathological changes seen in prion diseases.
Assuntos
Encéfalo/enzimologia , Hipocampo/enzimologia , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Scrapie/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/enzimologia , Astrócitos/patologia , Encéfalo/patologia , Citocromos c/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Hipocampo/patologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Neurônios/enzimologia , Neurônios/patologia , Óxido Nítrico Sintase Tipo III/genética , Scrapie/genética , Scrapie/patologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Regulação para CimaRESUMO
Apoptosis and autophagy are main mechanisms of neuronal death involved in prion diseases. Serum deprivation can induce both pathways to cell death in various types of cells. To investigate whether PrP(C) is involved in autophagy pathway, we analyzed the level of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, by monitoring the conversion from LC3-I into LC3-II in Zürich I Prnp(-/-) hippocampal neuronal cells. We found that the expression level of LC3-II was increased in Prnp(-/-) compared to wild-type cells under serum deprivation. In electron microscopy, increased accumulation of autophagosomes in Prnp(-/-) cells was correlated with the increase in levels of LC3-II. Interestingly, this up-regulated autophagic activity was retarded by the introduction of PrP(C) into Prnp(-/-) cells but not by the introduction of PrP(C) lacking octapeptide repeat region. Thus, the octapeptide repeat region of PrP(C) may play a pivotal role in the control of autophagy exhibited by PrP(C) in neuronal cells.
Assuntos
Autofagia , Neurônios/fisiologia , Príons/química , Príons/metabolismo , Análise de Variância , Animais , Caspase 3/metabolismo , Sobrevivência Celular/genética , Células Cultivadas , Hipocampo/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Microscopia Eletrônica de Transmissão/métodos , Microscopia Imunoeletrônica/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/ultraestrutura , Proteínas PrPC/metabolismo , Proteínas Priônicas , Príons/genética , Sequências Repetitivas de Aminoácidos , Soro/metabolismo , Fatores de Tempo , Transfecção , Regulação para Cima/fisiologiaRESUMO
HIV causes neurodegeneration and dementia in AIDS patients, but its function in milder cognitive impairments in virologically suppressed patients on antiretroviral therapy is unknown. Such patients are immunocompetent, have low peripheral and brain HIV burdens, and show minimal brain neuropathology. Using the model of HIV-related memory impairment in EcoHIV-infected conventional mice, we investigated the neurobiological and cognitive consequences of efficient EcoHIV expression in the mouse brain after intracerebral infection. HIV integrated and persisted in an expressed state in brain tissue, was detectable in brain monocytic cells, and caused neuroinflammatory responses and lasting spatial, working, and associative memory impairment. Systemic antiretroviral treatment prevented direct brain infection and memory dysfunction indicating the requirement for HIV expression in the brain for disease. Similarly inoculated murine leukemia virus used as a control replicated in mouse brain but not in monocytic cells and was cognitively benign, linking the disease to HIV-specific functions. Memory impairment correlated in real time with hippocampal dysfunction shown by defective long-term potentiation in hippocampal slices ex vivo and with diffuse synaptodendritic injury in the hippocampus reflected in significant reduction in microtubule-associated protein 2 and synapsin II staining. In contrast, there was no evidence of overt neuronal loss in this region as determined by neuron-specific nuclear protein quantification, TUNEL assay, and histological observations. Our results reveal a novel capacity of HIV to induce neuronal dysfunction and memory impairment independent of neurotoxicity, distinct from the neurotoxicity of HIV infection in dementia.IMPORTANCE HIV neuropathogenesis has been attributed in large measure to neurotoxicity of viral proteins and inflammatory factors produced by infected monocytic cells in the brain. We show here that HIV expression in mouse brain causes lasting memory impairment by a mechanism involving injury to hippocampal synaptodendritic arbors and neuronal function but not overt neuronal loss in the region. Our results mirror the observation of minimal neurodegeneration in cognitively impaired HIV patients on antiretroviral therapy and demonstrate that HIV is nonneurotoxic in certain brain abnormalities that it causes. If neurons comprising the cognition-related networks survive HIV insult, at least for some time, there is a window of opportunity for disease treatment.
Assuntos
Complexo AIDS Demência/fisiopatologia , Disfunção Cognitiva/fisiopatologia , Hipocampo/patologia , Hipocampo/virologia , Transtornos da Memória/complicações , Transtornos da Memória/fisiopatologia , Animais , Comportamento Animal , Modelos Animais de Doenças , Masculino , Camundongos , Carga ViralRESUMO
OBJECTIVE: Almost half of HIV-positive people on antiretroviral therapy have demonstrable mild neurocognitive impairment (HIV-NCI), even when virologically suppressed. Intranasal insulin therapy improves cognition in Alzheimer's disease and diabetes. Here we tested intranasal insulin therapy in a model of HIV-NCI in EcoHIV-infected conventional mice. DESIGN AND METHODS: Insulin pharmacokinetics following intranasal administration to mice was determined by ELISA. Mice were inoculated with EcoHIV to cause NCI; 23 days or 3 months after infection they were treated daily for 9 days with intranasal insulin (2.4âIU/mouse) and examined for NCI in behavioral tests and HIV burdens by quantitative PCR. Some animals were tested for hippocampal neuronal integrity by immunostaining and expression of neuronal function-related genes by real time-quantitative PCR. The effect of insulin treatment discontinuation on cognition and neuropathology was also examined. RESULTS: Intranasal insulin administration to mice resulted in µIU/ml levels of insulin in cerebrospinal fluid with a half-life of about 2âh, resembling pharmacokinetic parameters of patients receiving 40âIU. Intranasal insulin treatment starting 23 days or 3 months after infection completely reversed NCI in mice. Murine NCI correlated with reductions in hippocampal dendritic arbors and downregulation of neuronal function genes; intranasal insulin reversed these changes coincident with restoration of cognitive acuity, but they returned within 24âh of treatment cessation. Intranasal insulin treatment reduced brain HIV DNA when started 23 but not 90 days after infection. CONCLUSION: Our preclinical studies support the use of intranasal insulin administration for treatment of HIV-NCI and suggest that some dendritic injury in this condition is reversible.
Assuntos
Complexo AIDS Demência/tratamento farmacológico , Disfunção Cognitiva/tratamento farmacológico , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Administração Intranasal , Animais , Comportamento Animal , Modelos Animais de Doenças , Hipocampo/patologia , Hipoglicemiantes/farmacocinética , Imuno-Histoquímica , Insulina/farmacocinética , Camundongos Endogâmicos C57BL , Resultado do Tratamento , Carga ViralRESUMO
HIV-associated neurocognitive disorders (HAND) have been linked to dysregulation of glutamate metabolism in the central nervous system (CNS) culminating in elevated extracellular glutamate and disrupted glutamatergic neurotransmission. Increased glutamate synthesis via upregulation of glutaminase (GLS) activity in brain immune cells has been identified as one potential source of excess glutamate in HAND. However, direct evidence for this hypothesis in an animal model is lacking, and the viability of GLS as a drug target has not been explored. In this brief report, we demonstrate that GLS inhibition with the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) can reverse cognitive impairment in the EcoHIV-infected mouse model of HAND. However, due to peripheral toxicity DON is not amenable to clinical use in a chronic disease such as HAND. We thus tested JHU083, a novel, brain penetrant DON prodrug predicted to exhibit improved tolerability. Systemic administration of JHU083 reversed cognitive impairment in EcoHIV-infected mice similarly to DON, and simultaneously normalized EcoHIV-induced increases in cerebrospinal fluid (CSF) glutamate and GLS activity in microglia-enriched brain CD11b + cells without observed toxicity. These studies support the mechanistic involvement of elevated microglial GLS activity in HAND pathogenesis, and identify JHU083 as a potential treatment option. Graphical Abstract Please provide Graphical Abstract caption.Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders .
Assuntos
Complexo AIDS Demência , Compostos Azo/uso terapêutico , Caproatos/uso terapêutico , Transtornos Cognitivos/tratamento farmacológico , Glutamatos/biossíntese , Glutamina/antagonistas & inibidores , Pró-Fármacos/uso terapêutico , Animais , Compostos Azo/farmacocinética , Antígeno CD11b/análise , Caproatos/farmacocinética , Transtornos Cognitivos/líquido cefalorraquidiano , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/virologia , Condicionamento Clássico/efeitos dos fármacos , Medo , Glutamatos/líquido cefalorraquidiano , HIV-1/genética , HIV-1/patogenicidade , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/patogenicidade , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Memória de Curto Prazo/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Norleucina/análogos & derivados , Norleucina/uso terapêutico , Pró-Fármacos/farmacocinética , Vírus Reordenados/genética , Vírus Reordenados/patogenicidade , Aprendizagem Espacial/efeitos dos fármacosRESUMO
Previous studies have reported that various inbred SAM mouse strains differ markedly with regard to a variety of parameters, such as capacity for learning and memory, life spans and brain histopathology. A potential cause of differences seen in these strains may be based on the fact that some strains have a high concentration of infectious murine leukemia virus (MuLV) in the brain, whereas other strains have little or no virus. To elucidate the effect of a higher titer of endogenous retrovirus in astroglial cells of the brain, we established astroglial cell lines from SAMR1 and SAMP8 mice, which are, respectively, resistant and prone to deficit in learning and memory and shortened life span. MuLV-negative astroglial cell lines established from ICR mice served as controls. Comparison of these cell lines showed differences in: 1) levels of the capsid antigen CAgag in both cell lysates and culture media, 2) expression of genomic retroelements, 3) the number of virus particles, 4) titer of infectious virus, 5) morphology, 6) replication rate of cells in culture and final cell concentrations, 7) expression pattern of proinflammatory cytokine genes. The results show that the expression of MuLV is much higher in SAMP8 than SAMR1 astrocyte cultures and that there are physiological differences in astroglia from the 2 strains. These results raise the possibility that the distinct physiological differences between SAMP8 and SAMR1 are a function of activation of endogenous retrovirus.
Assuntos
Astrócitos/fisiologia , Astrócitos/virologia , Retrovirus Endógenos/isolamento & purificação , Retrovirus Endógenos/fisiologia , Animais , Proteínas do Capsídeo/biossíntese , Linhagem Celular , Citocinas/biossíntese , Perfilação da Expressão Gênica , Camundongos , Microscopia Eletrônica de Transmissão , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaio de Placa Viral , Vírion/isolamento & purificaçãoRESUMO
Considerable evidence suggests that oxidative stress may be involved in the pathogenesis of Transmissible Spongiform Encephalopathies (TSEs). To investigate the involvement of iron metabolism in TSEs, we examined the expression levels of iron regulatory proteins (IRPs), ferritins, and binding activities of IRPs to iron-responsive element (IRE) in scrapie-infected mice. We found that the IRPs-IRE-binding activities and ferritins were increased in the astrocytes of hippocampus and cerebral cortex in the brains of scrapie-infected mice. These results suggest that alteration of iron metabolism contributes to development of neurodegeneration and that some protective mechanisms against iron-induced oxidative damage may occur during the pathogenesis of TSEs.
Assuntos
Encéfalo/metabolismo , Ferritinas/metabolismo , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , Scrapie/metabolismo , Animais , Western Blotting , Ferritinas/genética , Expressão Gênica , Perfilação da Expressão Gênica , Imuno-Histoquímica , Ferro/metabolismo , Proteína 1 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/genética , Masculino , Camundongos , Reação em Cadeia da Polimerase , Scrapie/genéticaRESUMO
Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders (HAND). The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 14) attenuates glutamate synthesis in HIV-infected microglia/macrophages, offering therapeutic potential for HAND. We show that 14 prevents manifestation of spatial memory deficits in chimeric EcoHIV-infected mice, a model of HAND. 14 is not clinically available, however, because its development was hampered by peripheral toxicities. We describe the synthesis of several substituted N-(pivaloyloxy)alkoxy-carbonyl prodrugs of 14 designed to circulate inert in plasma and be taken up and biotransformed to 14 in the brain. The lead prodrug, isopropyl 6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (13d), was stable in swine and human plasma but liberated 14 in swine brain homogenate. When dosed systemically in swine, 13d provided a 15-fold enhanced CSF-to-plasma ratio and a 9-fold enhanced brain-to-plasma ratio relative to 14, opening a possible clinical path for the treatment of HAND.
Assuntos
Aminocaproatos/farmacologia , Compostos Azo/farmacologia , Diazo-Oxo-Norleucina/farmacologia , Transtornos Neurocognitivos/tratamento farmacológico , Nootrópicos/farmacologia , Pró-Fármacos/farmacologia , Aminocaproatos/administração & dosagem , Aminocaproatos/síntese química , Animais , Compostos Azo/administração & dosagem , Compostos Azo/síntese química , Sangue/metabolismo , Encéfalo/metabolismo , Diazo-Oxo-Norleucina/administração & dosagem , Estabilidade de Medicamentos , Feminino , Ácido Glutâmico/metabolismo , Glutaminase/antagonistas & inibidores , Infecções por HIV/complicações , Humanos , Masculino , Camundongos Endogâmicos C57BL , Transtornos Neurocognitivos/etiologia , Nootrópicos/administração & dosagem , Nootrópicos/síntese química , Pró-Fármacos/administração & dosagem , Pró-Fármacos/síntese química , Suínos , Carga Viral/efeitos dos fármacosRESUMO
Prion diseases are infectious and fatal neurodegenerative diseases which require the cellular prion protein, PrPC, for development of diseases. The current study shows that the PrPC augments infectivity and plaque formation of a mouse endogenous retrovirus, MuLV. We have established four neuronal cell lines expressing mouse PrPC, PrP+/+; two express wild type PrPC (MoPrPwild) and the other two express mutant PrPC (MoPrPmut). Infection of neuronal cells from various PrP+/+ and PrP-/- (MoPrPKO) lines with MuLV yielded at least three times as many plaques in PrP+/+ than in PrP-/-. Furthermore, among the four PrP+/+ lines, one mutant line, P101L, had at least 2.5 times as many plaques as the other three PrP+/+ lines. Plaques in P101L were four times larger than those in other PrP+/+ lines. Colocalization of PrP and CAgag was seen in MuLV-infected PrP+/+ cells. In the PrP-MuLV interaction, the involvement of galectin-3 and -6 was observed by immunoprecipitation with antibody to PrPC. These results suggest that PrPC combined with galectin-3 and -6 can act as a receptor for MuLV. P101L, the disease form of mutant PrPC results suggest the genetic mutant form of PrPC may be more susceptible to viral infection.
Assuntos
Galectina 3/metabolismo , Galectinas/metabolismo , Vírus da Leucemia Murina/crescimento & desenvolvimento , Neurônios/metabolismo , Proteínas PrPC/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/metabolismo , Astrócitos/virologia , Western Blotting , Linhagem Celular , Células Cultivadas , Retrovirus Endógenos/crescimento & desenvolvimento , Retrovirus Endógenos/fisiologia , Galectina 3/genética , Galectinas/genética , Hipocampo/citologia , Hipocampo/virologia , Interações Hospedeiro-Patógeno , Vírus da Leucemia Murina/fisiologia , Camundongos Knockout , Microscopia Confocal , Neurônios/citologia , Neurônios/virologia , Proteínas PrPC/genética , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Prions have been extensively studied since they represent a new class of infectious agents, the pathogenic prion protein (PrPSc). However, a central question on the physiological function of the normal prion protein (PrPC) remains unresolved. A cell model which was previously established from Rikn mice (PrP-/-) remains problematic because of its ectopic expression of the doppel (Dpl) which may have a neurotoxic effect. Here we established neuronal cell lines from Zürich I (PrP-/-) which do not express Dpl protein and ICR mice (PrP+/+) by transfecting with plasmid encoding for the large T antigen of SV40. The transformed cells have shown neuronal characteristics and, thus, these cell lines may provide a useful model to explore the function of neuronal PrPC.
Assuntos
Hipocampo/citologia , Neurônios/metabolismo , Proteínas PrPC/metabolismo , Príons/metabolismo , Animais , Western Blotting/métodos , Contagem de Células/métodos , Ciclo Celular/fisiologia , Linhagem Celular Transformada/metabolismo , Tamanho Celular , Células Cultivadas , Embrião de Mamíferos , Imunofluorescência/métodos , Proteínas Ligadas por GPI , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas PrPC/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de TempoRESUMO
Prion diseases are transmissible neurodegenerative disorders that are invariably fatal in humans and animals. Although the nature of the infectious agent and pathogenic mechanisms of prion diseases are not clear, it has been reported that prion diseases may be associated with aberrant metabolism of cellular prion protein (PrP(C)). In various reports, it has been postulated that PrP(C) may be involved in one or more of the following: neurotransmitter metabolism, cell adhesion, signal transduction, copper metabolism, antioxidant activity or programmed cell death. Despite suggestive results supporting each of these mechanisms, the physiological function(s) of PrP(C) is not known. To investigate whether PrP(C) can prevent apoptotic cell death in prion diseases, we established the cell lines stably expressing PrP(C) from PrP knockout (PrP(-/-)) neuronal cells and examined the role of PrP(C) under apoptosis and/or serum-deprived condition. We found that PrP(-/-) cells were vulnerable to apoptotic cell death and that this vulnerability was rescued by the expression of PrP(C). The expression levels of apoptosis-related proteins including p53, Bax, caspase-3, poly(ADP-ribose) polymerase (PARP) and cytochrome c were significantly increased in PrP(-/-) cells. In addition, Ca(2+) levels of mitochondria were increased, whereas mitochondrial membrane potentials were decreased in PrP(-/-) cells. These results strongly suggest that PrP(C) may play a central role as an effective anti-apoptotic protein through caspase-dependent apoptotic pathways in mitochondria, supporting the concept that disruption of PrP(C) and consequent reduction of anti-apoptotic capacity of PrP(C) may be one of the pathogenic mechanisms of prion diseases.
Assuntos
Apoptose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Proteínas PrPC/farmacologia , Soro/metabolismo , Animais , Western Blotting/métodos , Cálcio/metabolismo , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Fragmentação do DNA/fisiologia , Relação Dose-Resposta a Droga , Fluoresceínas/metabolismo , Hipocampo/citologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , Neurônios/metabolismo , Reação em Cadeia da Polimerase/métodos , Proteínas PrPC/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Inanição , Fatores de Tempo , Transfecção/métodos , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2RESUMO
The polyoma group of viruses, including SV40, is known to be oncogenic in certain species. Here we report for the first time naturally occurring, immortalized tumor cells from a patient with glioblastoma multiforme (GBM); the cells were shown to be oligodendroglia; cells had developed remarkable chromosomal changes and were positive for SV40 T antigen. Therefore, we postulated that the main cause of immortalization of these cells was the expression of SV40 T antigen gene and protein. Since the cells are naturally generated, they will provide a useful model to study the function of oligodendroglial cells and the development of GBM.